Cancers with germline or somatic mutations of BRCA1/2 have compromised DNA repair pathway, homologous recombination (HR) deficiency. These cancers have a unique response to PARP inhibitors and consequently BRCA1/2 mutations are markers to determine treatment options for breast and ovarian cancers. There is an urgent need to identify additional markers in all cancers that indicate deficiency in HR and consequently application of PARP inhibitors for these cancers. The DNA homologous repair pathway requires many proteins and enzymes to coordinate the repair of DNA double-stranded breaks. Among them, a DNA polymerase is essential for the key step of D-loop extension. In this application, we hypothesize that a specific form of DNA polymerase ?, Pol ?4, is required for HR. Thus, cancer cells without Pol ?4 are HR deficient and sensitive to PARP inhibition. The hypothesis is based on our work in characterization of the enzymatic properties of Pol ?4, especially the ability in strand displacement synthesis. Indeed, it has been shown that the majority of small cell lung cancer (SCLC) cells lack Pol ?4, which is also supported by our preliminary data. The main goal of this project is to test our central hypothesis that Pol ?4 is the key form of polymerase that can perform D-loop extension to facilitate homologous recombination repair of DNA double-stranded breaks. Cancer cells deficient in Pol ?4 are HR deficient and amendable to PARP inhibition. Specifically, this project will 1) Establish that Pol ?4 is required for homologous recombination and the consequences of Pol ?4-deficiency to genomic stability; 2) Characterize the unique response of Pol ?4 deficient cells to chemotherapeutic agents and PARP inhibitors; 3) Elucidate the regulation of D-loop extension by Pol d4 by 3?-5? helicases, including BLM, WRN and DHX9. The proposed research is innovative as we employ multidisciplinary approaches that include cellular studies and enzymatic characterization to elucidate the underlying mechanisms of Pol ?4 function in D-loop extension. The insight from how changes in subunit composition of Pol ? affect its function in DNA replication and repair is innovative. Completion of this proposal will be a significant contribution to our understanding of how DNA damages lead to carcinogenesis due to alteration of Pol ? quaternary structure. The results in this proposal can potentially change clinical practice in cancer treatment and define a new class of cancers that can be treated with PARP inhibitors.